The p75 neurotrophin receptor plays a key role in regulating the delicate balance between neuronal survival and death during the development of the mammalian nervous system. In addition, it has been implicated in a number of neuropathologies involving abnormal apoptosis, such as Alzheimer's disease, spinal cord lesion and excitotoxic injury. Like other members of the TNF receptor superfamily, p75 activates a bifurcating signal that promotes both cell survival and cell death. How these pathways are activated and differentially regulated to determine the ultimate fate of a cell is not well understood. The overall objective of this proposal is to elucidate the molecular mechanisms by which the p75 neurotrophin receptor mediates cell death. We previously demonstrated that TRAF6 and NRIF are two receptor interacting proteins essential for p75- mediated apoptosis. TRAF6 is an E3 ubiquitin ligase known to regulate activation of NF-kB and JNK by many members of the TNF and Toll-like receptor superfamilies. In contrast, NRIF is a novel DMA binding protein that associates with the intracellular domain (ICD) of p75, thus raising the question as to how this interactor gets to the nucleus and whether p75 regulates NRIF's transcriptional activity. Our preliminary data indicate that NRIF is ubiquitinated by TRAF6 and this promotes its nuclear translocation. In addition, p75 was recently shown to undergo intramembrane proteolysis by gamma-secretase, leading to the release of the ICD. How this processing affects signaling is not clear;however, our preliminary data indicate that this is required for the receptor to induce apoptosis. A similar cleavage has been shown to mediate nuclear signaling for a number of transmembrane proteins (e.g. Notch and APR). Therefore, we hypothesize that p75 cleavage releases NRIF, which is targeted to the nucleus by TRAF6-mediated ubiquitination, resulting in the regulation of gene transcription and subsequently apoptosis. To test this hypothesis we propose to determine (1) the relationship between p75 cleavage and its signal transduction, (2) the role of receptor proteolysis in the nuclear shuttling of NRIF, (3) the functional consequence of NRIF ubiquitination, and (4) the change in NRIF's transcriptional activity in response to p75 activation. Given the ever-growing number of pathological conditions where p75 has been implicated, it is imperative that we begin to understand how this receptor mediates cell death in order to develop intervention strategies.